Hyperlipidaemia attributes to around one third of Cardiovascular Diseases (CVD) worldwide [1]. Clinically, it is mainly defined by elevated TC and/or Low-Density Lipoprotein Cholesterol (LDL-C), but the definition is also often extended to include non optimal levels of High Density Lipoprotein Cholesterol (HDL-C), TG, apolipoprotein B [2-4]. Lipid disorders can be mainly classified into primary or secondary to some underlying cause like environmental factors (diet rich in saturated fat and trans fat or a sedentary lifestyle), diseases (type 2 diabetes, chronic kidney disease, hypothyroidism, etc.,) and even medications (thiazide, β-blockers, progestins, anabolic steroids etc.,) [5,6].
There are various evidences in support of the fact that successful treatment of dyslipidaemia reduces morbidity and mortality from CVD. It is therefore desired to make a comprehensive strategy which includes various ways to control lipid levels and to also address associated metabolic abnormalities and modifiable risk factors such as obesity, hypertension, diabetes, and cigarette smoking.
GS an indigenous herb belongs to the family Asclepiadaceae. The plant is also known as Gurmar in Hindi which means “destroyer of sugar” [10]. Powdered extract of its leaves is known to possess various properties: astringent, anti-inflammatory, diuretic, digestive, liver tonic, emetic, stimulant, antihelminthic, laxative, expectorant, antipyretic and diuretic. Various animal and human studies have shown the anti-diabetic properties of GS [11-13].
Some of the studies have been conducted in the past to explore the hypolipidaemic activity of this plant. But any conclusive inferences could not be drawn from those as some favoured the hypolipidaemic activity while others showed its absence. The various animal models that had been used were streptozocin induced diabetes and alloxan induced diabetic models. Present study was designed to explore and support the hypolipidaemic property of GS using High Fat Diet (HFD) rodent model and to further compare it with the standard drug Atorvastatin.
Materials and Methods
Before commencing this animal experimental work ethical approval was taken from Institutional Animal Ethics Committee (IAEC), King George’s Medical University, Lucknow, Uttar Pradesh, India (Research project No. 69/IAEC/2015).
A total of 30 adult healthy female wistar rats, weighing 150-200 g, were procured from the Indian Institute of Toxicology Research (IITR), Lucknow. They were kept at Institutional animal house of King George’s Medical University, Lucknow, Uttar Pradesh, India. Standard conditions of temperature (25±2ºC), humidity (55±05%) and proper light dark cycle controlled environment were maintained throughout the work. Rats were given pellet diet and free access to drinking water.
After a period of acclimatisation for seven days, they were randomly divided into five groups with six rats in each group. On day 0, blood sample was collected for the estimation of lipid profile. During phase I (for four weeks) of the study, Group 1 (Normal Control) was given normal chow while Group 2 to 5 were kept on a HFD. HFD was provided by Bharat Science Solution Company, Lok Nagar, Unnao, Uttar Pradesh. It contained crude fat 25%, crude protein 18%, carbohydrate 44%, fiber 13%, moisture 8%, vitamins, minerals and other ingredients in appropriate quantity.
During phase II of the study (from fifth week to eighth week), Group 1 was continued on normal chow diet while Group 2 was continued on HFD. Other groups were given HFD along with respective drugs. Group 3 was given GS (100 mg/kg), Group 4 was on GS (200 mg/kg), Group 5 was given Atorvastatin (10 mg/kg). The drugs were given as a suspension in distilled water by oral route.
Test Drug
GS: Powdered extract was obtained from Ekgaon Company, New Delhi. The drug was administered in a dose of 100 and 200 mg/kg [14].
Standard Drug
Atorvastatin was given in a dose of 10 mg/kg [15].
Measurement of Body Weight
The body weight of rats was measured using digital weighing machine.
Collection of Blood Sample
By retro-orbital route after anaesthetising animals by using Pentobarbital (50 mg/kg, intraperitoneal).
Estimation of Plasma Lipid Profile
For estimation of TC, TG, HDL-C, Selectra E, a fully automated clinical chemistry analyser, manufactured by Vital scientific N.V. Netherlands and commercial kits based on enzymatic assay method (manufactured by Elitech Clinical Systems, France) were used.
Estimation of LDL-C and VLDL-C was done indirectly by using the formula:
LDL = TC – (HDL + TG/5) VLDL = TG/5
Statistical Analysis
All the analyses was performed by SPSS Statistics 20 (IBM software). Data was recorded as mean±SEM (Standard error of mean). Paired t-test was used to compare a single group at different point of time. To make intergroup comparison, one way ANOVA followed by Dunnett’s post hoc test was used. A p-value<0.05 was considered statistically significant.
Results
On Day 0 all the rats were weighed and their blood sample was taken for lipid profile estimation. On applying ANOVA it was found that all the groups were comparable in terms of body weight and lipid profile with no significant difference among the groups [Table/Fig-1].
Comparison of different groups by ANOVA at day 0.
| Parameters | F-value | p-value |
|---|
| Body weight | 0.266 | 0.897 |
| TC | 0.337 | 0.850 |
| TG | 0.303 | 0.873 |
| HDL | 0.124 | 0.972 |
| VLDL | 0.303 | 0.873 |
| LDL | 1.036 | 0.408 |
On feeding rats of Group 2 to 5 with HFD for four weeks, their body weight increased significantly and there were significant changes in the lipid profile as calculated by paired t-test. TC, TG, VLDL and LDL increased significantly from their respective values at day 0 while HDL fell significantly [Table/Fig-2].
Body weight (g) of all the Groups (mean ± SEM, n=6).
| GROUPS | DAY 0 | WEEK 4 | week 8 | % change at week 8 as compared to group 2 |
|---|
| Group 1 | 196.6±3.11 | 198.2±4.2 | 203.3±2.7 | 32.7 |
| Group 2 | 197.1±7.5 | 292.0±3.7* | 331.8±5.0#$ | - |
| Group 3 | 188.2±8.6 | 284.9±7.4* | 222.7±3.3#$ | 32.9 |
| Group 4 | 188.4±11.7 | 283.8±9.2* | 205.5±9.0#$ | 38.1 |
| Group 5 | 189.9±9.4 | 285.5±9.0* | 300.7±7.7#$ | 9.37 |
Significant as compared to baseline (Day 0)
Significant as compared to week 4
Significant as compared to baseline (Day 0)
Effect on Body Weight
During phase II of the study (between fifth and eighth week) all the groups were given their respective diet and drugs. In Group 2 (on HFD only) and Group 5 (Atorvastatin) body weight increased further while body weight reduced significantly (p-value <0.01) in Group 3 and 4. GS (100 mg/kg) and GS (200 mg/kg) caused 32.9% and 38.1% reduction in body weight when compared to the Group 2 (Disease control/HFD group).
At the end of eighth week Dunnett’s post hoc test with Group 2 as comparison group showed that test drugs reduced body weight significantly [Table/Fig-3]. When compared to Group 1 no significant difference was found in Group 3 and 4.
ANOVA followed by Dunnett’s post hoc test (comparison Groups-2 and 5) at the end of eighth week for body weight.
| Y | X |
|---|
| group 1 (Normal control) | Group 2 (Disease Control) | Group 5 (Atorvastatin Control) |
|---|
| Mean Difference (Y-Group 1) | p-value | Mean Difference (Y-Group 2) | p-value | Mean Difference (Y-Group 5) | p-value |
|---|
| Group 1 | - | - | -128.5* | <0.01 | -97.4* | <0.01 |
| Group 2 | 128.5* | <0.01 | | | 31.1* | <0.01 |
| Group 3 | 19.4 | 0.10 | -109.1* | <0.01 | -78.0* | <0.01 |
| Group 4 | 2.2 | 1.00 | -126.3* | <0.01 | -95.3* | <0.01 |
| Group 5 | 97.4* | <0.01 | -31.1* | <0.01 | - | - |
Significant
Effect on TC
On feeding Groups 2 to 5 with HFD, the value of TC increased significantly after four weeks while there was no significant change in Group 1 [Table/Fig-4]. Further on giving drug treatment, the value of TC reduced significantly (p-value< 0.01) in all the drug groups (Group 3 to 5). Highest reduction (40.2%) in the value of TC occurred in Group 5 (Atorvastatin group) followed by 21.8% in Group 4 (GS-200 mg/kg) and 15.8% in Group 3 (GS-100 mg/kg).
Pre and post treatment Total Cholesterol (mg/dl) of all the Groups (mean ± SEM, n=6).
| GROUPS | DAY 0 | WEEK 4 | WEEK 8 | % change at week 8 as compared to Group 2 |
|---|
| Group 1 | 81.3±2.5 | 81.3±2.8 | 81.2±2.1 | 52.2 |
| Group 2 | 80.9±2.4 | 152.6±2.7* | 170.0±3.0#$ | - |
| Group 3 | 78.5±3.1 | 155.4±2.9* | 143.1±2.6#$ | 15.8 |
| Group 4 | 83.8±3.2 | 154.8±3.3* | 133.0±2.6#$ | 21.8 |
| Group 5 | 80.4±4.6 | 155.5±2.6* | 101.6±2.2#$ | 40.2 |
Significant as compared to baseline (Day 0)
Significant as compared to week 4
Significant as compared to baseline (Day 0)
On comparing with disease control (by Dunnett’s post hoc test), TC decreased significantly in all the drug groups. But when compared to Atorvastatin control, significant difference was found. This showed that the test drug reduced the value of TC but it was not as much as that by Atorvastatin [Table/Fig-5].
ANOVA followed by Dunnett’s post hoc test (comparison Groups-2 and 5) at the end of eighth week for Total Cholesterol.
| Y | X |
|---|
| Group 1 (Normal Control) | Group 2 (Disease Control) | Group 5 (Atorvastatin Control) |
|---|
| Mean Difference (Y-Group 1) | p-value | Mean Difference (Y-Group 2) | p-value | Mean Difference (Y-Group 5) | p-value |
|---|
| Group 1 | - | - | -88.9* | <0.01 | -20.4* | <0.01 |
| Group 2 | 88.9* | <0.01 | | | 68.4* | <0.01 |
| Group 3 | 62.0* | <0.01 | -26.9* | <0.01 | 41.6* | <0.01 |
| Group 4 | 51.8* | <0.01 | -37.1* | <0.01 | 31.4* | <0.01 |
| Group 5 | 20.4* | <0.01 | -68.4* | <0.01 | - | - |
Significant.
Effect on Triglycerides
After giving HFD for four weeks to Group 2 to 5, the value of TG increased significantly. Introduction of drug treatment after fourth week lead to decrease in the value of TG in all the groups on drug (Group 3 to 5). Atorvastatin (Group 5) lead to highest reduction (37.4%) in TG followed by GS-200 mg/kg in Group 4 by 23.6% and GS-100 mg/kg in Group 3 by 18.0% [Table/Fig-6].
Pre and post treatment TG (mg/dl) of all the groups (mean±SEM, n=6).
| GROUPS | DAY 0 | WEEK 4 | WEEK 8 | % change at week 8 as compared to Group 2 |
|---|
| Group 1 | 67.2±3.3 | 69.5±3.2 | 67.6±1.7 | 55.5 |
| Group 2 | 69.3±3.0 | 138.3±1.5* | 152.0±1.8#$ | - |
| Group 3 | 68.7±4.1 | 138.6±2.3* | 124.7±2.4#$ | 18.0 |
| Group 4 | 72.8±3.7 | 138.6±2.4* | 116.2±1.9#$ | 23.6 |
| Group 5 | 69.6±4.5 | 138.4±2.6* | 95.2±2.9#$ | 37.4 |
Significant as compared to baseline (Day 0)
Significant as compared to week 4
Significant as compared to baseline (Day 0)
On applying Dunnett’s post hoc test at the end of eighth week with Group 2 as comparison group, it was found that the value of TG fell significantly in all the drug groups as compared to disease control (Group 2). On comparing with Group 5 (Atorvastatin group), significant difference was found [Table/Fig-7]. Hence it can be said that although test drugs reduced the value of TG significantly yet it was found inferior to Atorvastatin.
ANOVA followed by Dunnett’s post hoc test (comparison Groups-2 and 5) at the end of eighth week for Triglycerides.
| Y | X |
|---|
| Group 1 (Normal Control) | Group 2 (Disease Control) | Group 5 (Atorvastatin Control) |
|---|
| Mean Difference (Y-Group 1) | p-value | Mean Difference (Y-Group 2) | p-value | Mean Difference (Y-Group 5) | p-value |
|---|
| Group 1 | - | - | -84.4* | <0.01 | -27.6* | <0.01 |
| Group 2 | 84.4* | <0.01 | | | 56.8* | <0.01 |
| Group 3 | 57.0* | <0.01 | -27.4* | <0.01 | 29.4* | <0.01 |
| Group 4 | 48.6* | <0.01 | -35.8* | <0.01 | 21.0* | <0.01 |
| Group 5 | 27.6* | <0.01 | -56.8* | <0.01 | - | - |
Significant
Effect on HDL-C
HFD for four weeks caused HDL values to fall significantly in Groups 2 to 5 as shown in [Table/Fig-8]. After fourth week drug treatment was started. This lead to increase in the value of HFD in all the drug groups while it further decreased in HFD group (Group 2). Highest increase in the value of HDL occurred in Group 5 (76.3%) followed by in Group 4 (39.4%) and in Group 3 (30.3%) as compared to disease control group (Group 2).
Pre and post treatment HDL (mg/dl) of all the groups (Mean±SEM, n=6).
| GROUPS | DAY 0 | WEEK 4 | WEEK 8 | % change at week 8 as compared to Group 2 |
|---|
| Group 1 | 33.4±1.0 | 33.8±1.3 | 32.8±1.1 | 65.7 |
| Group 2 | 33.2±1.3 | 22.4±1.0* | 19.8±0.9#$ | - |
| Group 3 | 32.5±1.2 | 22.8±1.1* | 25.8±0.9#$ | 30.3 |
| Group 4 | 33.0±1.6 | 23.1±1.1* | 27.6±1.0#$ | 39.4 |
| Group 5 | 33.8±1.5 | 23.6±0.9* | 34.9±1.3#$ | 76.3 |
Significant as compared to baseline (Day 0)
Significant as compared to week 4
Significant as compared to baseline (Day 0)
Comparing with disease control (Group 2), HDL increased in all the drug groups but this effect was found inferior to the Atorvastatin as shown by Dunnett’s post hoc test [Table/Fig-9].
ANOVA followed by Dunnett’s post-hoc test (comparison Groups-2 and 5) at the end of eighth week for HDL.
| Y | X |
|---|
| Group 1 (Normal Control) | Group 2 (Disease Control) | Group 5 (Atorvastatin Control) |
|---|
| Mean Difference (Y-Group 1) | p-value | Mean Difference (Y-Group 2) | p-value | Mean Difference (Y-Group 5) | p-value |
|---|
| Group 1 | - | - | 13.0* | <0.001 | -2.1 | 0.447 |
| Group 2 | -13.0* | <0.01 | | | -15.0* | <0.01 |
| Group 3 | -7.0* | <0.01 | 6.0* | 0.002 | -9.1* | <0.01 |
| Group 4 | -5.2* | <0.01 | 7.7* | <0.001 | -7.3* | <0.01 |
| Group 5 | 2.1 | 0.447 | 15.0* | <0.001 | - | - |
*Significant
Effect on VLDL
Feeding rats with HFD lead to increase in the value of VLDL in Groups 2 to 5. On starting drug treatment after fourth week, value of VLDL decreased significantly in Groups 3 to 5 while it further increased in HFD group (Group 2). Atorvastatin lead to highest reduction (37.5%) in VLDL value followed by GS-200 mg/kg (23.7%) and GS-100 mg/kg (18.1%) [Table/Fig-10].
Pre and post treatment VLDL (mg/dl) of all the groups (Mean±SEM, n=6).
| GROUPS | DAY 0 | WEEK 4 | WEEK 8 | % change at week 8 as compared to Group 5 |
|---|
| Group 1 | 13.4±0.7 | 13.9±0.6 | 13.6±0.3 | 55.6 |
| Group 2 | 13.9±0.6 | 27.7±0.3* | 30.4±0.4#$ | - |
| Group 3 | 13.7±0.8 | 27.7±0.5* | 24.9±0.7#$ | 18.1 |
| Group 4 | 14.6±0.7 | 27.7±0.5* | 23.2±0.4#$ | 23.7 |
| Group 5 | 13.9±0.9 | 27.7±0.5* | 19.1±0.6#$ | 37.5 |
Significant as compared to baseline (Day 0)
Significant as compared to week 4
Significant as compared to baseline (Day 0)
Dunnett’s post hoc test with Group 2 as comparison group showed that all the drugs reduced the value of VLDL significantly but when compared to Group 5, VLDL reducing effect of test drug was found inferior to Atorvastatin [Table/Fig-11].
ANOVA followed by Dunnett’s post hoc test (comparison Groups-2 and 5) at the end of eighth week for VLDL.
| Y | X |
|---|
| Group 1 (Normal Control) | group 2 (Disease control) | Group 5 (Atorvastatin Control) |
|---|
| Mean Difference (Y-Group 1) | p-value | Mean Difference (Y-Group 2) | p-value | Mean Difference (Y-Group 5) | p-value |
|---|
| Group 1 | - | - | -16.9* | <0.01 | -5.5* | <0.01 |
| Group 2 | 16.9* | <0.01 | | | 11.4* | <0.01 |
| Group 3 | 11.4* | <0.01 | -5.5* | <0.01 | 5.9* | <0.01 |
| Group 4 | 9.7* | <0.01 | -7.2* | <0.01 | 4.2* | <0.01 |
| Group 5 | 5.5* | <0.01 | -11.45* | <0.01 | - | - |
Significant.
Effect on LDL
HFD for four weeks lead to derangement of lipid profile. Value of LDL increased in all the groups on HFD. After starting drugs, value of LDL decreased in Group 3 to 5. Maximum reduction occurred in Group 5 on Atorvastatin (60.2%) followed by Group 4 (31.4%) and Group 3 (22.9%) [Table/Fig-12].
Pre and post treatment LDL (mg/dl) of all the groups (Mean±SEM, n=6).
| GROUPS | DAY 0 | WEEK 4 | WEEK 8 | % change at week 8 as compared to Group 2 |
|---|
| Group 1 | 34.5±1.5 | 33.5±1.9 | 34.9±1.9 | 70.9 |
| Group 2 | 33.8±1.0 | 102.6±2.0* | 119.8±2.2#$ | - |
| Group 3 | 32.3±1.6 | 104.9±1.8* | 92.4±1.7#$ | 22.9 |
| Group 4 | 36.3±1.3 | 104.0±2.6* | 82.2±1.8#$ | 31.4 |
| Group 5 | 32.7±2.2 | 104.2±1.9* | 47.7±1.3#$ | 60.2 |
Significant as compared to baseline (Day 0)
Significant as compared to week 4
Significant as compared to baseline (Day 0)
When compared to Group 2 (by Dunnett’s post hoc test at the end of eighth week) it was found that all the drugs reduced LDL values significantly. On considering Group 5 as comparison group, significant difference was found. Thus the LDL reducing effect of test drugs was found inferior to Atorvastatin (10 mg/kg) [Table/Fig-13].
ANOVA followed by Dunnett’s post hoc test (comparison Groups-2 and 5) at the end of eighth week for LDL.
| Y | X |
|---|
| Group 1 (Normal Control) | Group 2 (Disease Control) | Group 5 (Atorvastatin Control) |
|---|
| Mean Difference (Y-Group 1) | p-value | Mean Difference (Y-Group 2) | p-value | Mean Difference (Y-Group 5) | p-value |
|---|
| Group 1 | - | - | -84.9* | <0.01 | -12.8* | <0.01 |
| Group 2 | 84.9* | <0.01 | - | - | 72.1* | <0.01 |
| Group 3 | 57.5* | <0.01 | -27.3* | <0.01 | 44.7* | <0.01 |
| Group 4 | 47.3* | <0.01 | -37.6* | <0.01 | 34.5* | <0.01 |
| Group 5 | 12.8* | <0.01 | -72.1* | <0.01 | - | - |
Significant.
Discussion
Diet rich in fats (HFD) given to rats for four weeks induced significant weight gain along with derangement of lipid profile. TC, TG, VLDL and LDL values increased significantly while that of HDL fell significantly. These findings suggest that the rats fed with HFD can act as a model of obesity and dyslipidaemia.
Powdered extract of GS was given to rats in two doses of 100 mg/kg and 200 mg/kg and effect was compared to that of Atorvastatin. GS at both the doses reduced TC but effect was inferior to Atorvastatin. Value of TG also decreased significantly but not equivalent to that of standard drug. The extract of Gymnema also improved the value of HDL-C significantly yet effect was lower as compared to Atorvastatin. GS also decreased the value of LDL and VLDL significantly but the effect was inferior to the standard drug Atorvastatin. The findings of present study are in accordance with some of the previous studies [16]. Mall GK et al., investigated the antidiabetic and hypolipidaemic activity of aqueous leaf extract in Alloxan induced diabetic rats at variable dosage [12].
Extract of GS are rich in phytochemicals, gymnemic acids and gurmarin are the active one [17]. Gurmarin is known to block the ability to taste sweet and bitter flavors thus reduces craving for food [18]. Palatal taste buds are innervated by greater petrosal nerve; gurmarin acts on it and decreases the phasic response to sugars [19]. Along with glucose it also decreases the intestinal absorbtion of histidine and oleic acids [20]. Gymnemagenin present in the leaf extracts increases the fecal excretion of cholesterol, neutral steroids and bile acids [21].
Aziza AM et al., demonstrated that treatment of streptozocin induced diabetic rats with leaf extract of GS significantly reduced the blood glucose, TC, LDL-C levels and increased insulin and HDL-C levels [22]. Rachh PR et al., showed that treating rats with leaf extract of GS increased the value of HDL and reduced TC, TG, LDL and VLDL [14]. The present study shows that leaf extract of GS possess significant hypolipidaemic action and strengthens the findings of some of the studies conducted to explore the lipid lowering action of GS.
Limitation
The present work is an animal experimental study to strengthen the effects of GS on lipid profile in animals. The results are limited to only two doses. This study has limitation of proper dose response relationship of GS on lipid profile. The use of only single model is another limitation. The number of animals in each group was six which may be another constraint.
Conclusion
The findings of the present study affirm that GS at both the doses (100 and 200 mg/kg) has a significant hypolipidaemic action but the effects were inferior to the standard drug Atorvastatin. It can be considered to use the active constituents of this plant as an add on therapy for hyperlipidaemia. Herbal drugs are considered safer and cheaper alternatives. Hence it would be to prudent to promote more studies on higher animals and humans so as to affirm the findings of present study.
*Significant as compared to baseline (Day 0)#Significant as compared to week 4$Significant as compared to baseline (Day 0)